Protein ubiquitination is a versatile posttranslational modification with roles in protein degradation, cell signaling, intracellular trafficking and the DNA damage response (Chen and Sun, 2009; Komander, 2009). Ubiquitin polymers are linked through one of seven internal lysine (K) residues or through the N-terminal amino group. Importantly, the type of ubiquitin linkage determines the functional outcome of the modification (Komander, 2009). The best-studied ubiquitin polymers, K48- and K63-linked chains, have degradative and non-degradative roles, respectively (Chen and Sun, 2009; Hershko and Ciechanover, 1998). However, recent data has revealed an unexpected high abundance of so-called atypical ubiquitin chains; for example, K11 linkages have been found to be as abundant as K48-linkages in S. cerevisiae (Peng et al., 2003; Xu et al., 2009).
Polyubiquitin chains are assembled on substrates through the concerted action of a three-step enzymatic cascade, involving an E1 ubiquitin activating enzyme, an E2 ubiquitin conjugating enzyme, and E3 ubiquitin ligases (Dye and Schulman, 2007). While E3 ligases attach polyubiquitin chains to a target and thus confer substrate specificity, E2 enzymes are thought to determine the type of chain linkage in polyubiquitin chains. K48- and K63-specific E2 enzymes have been identified (Chen and Pickart, 1990; Hofmann and Pickart, 1999), which allowed structural analysis of these chain types as well as a detailed understanding of specificity of ubiquitin binding domains (UBDs) and deubiquitinases (DUBs) (reviewed in Komander, 2009). This information is currently lacking for atypical ubiquitin chains.
Several recent reports have implicated K11-linked ubiquitin chains in distinct biological processes. Early data indicated that K11-linked chains are proteasomal degradation signals (Baboshina and Haas, 1996). An E2 enzyme, UBE2S/E2-EPF, was identified that assembled K11 linkages in vitro (Baboshina and Haas, 1996). The human anaphase promoting complex (APC/C) was found to assemble K11 linkages using the E2 enzyme UBE2C/UbcH10, on proteins that need to be degraded for cell cycle progression (Jin et al., 2008). A yeast proteomics study, apart from having revealed the high abundance of K11 linkages, also implicated this chain type with endoplasmic reticulum-associated degradation (ERAD), and identified yeast Ubc6 as an E2 enzyme involved in synthesis of K11-linked chains (Xu et al., 2009). In mammalian cells, K11 linkages were found to be enriched in UBA/UBX protein complexes, which interact with the key ERAD regulator p97/cdc48 (Alexandru et al., 2008). Hence, K11-linked chains seem to regulate numerous important cellular processes, and may act as a distinct proteasomal degradation signal. However, cellular mechanisms of assembly and disassembly of K11 linkages, as well as structural determinants for K11 linkage recognition, are unknown.
The structure of E2 enzymes is well characterised. All E2 enzymes comprise a conserved domain of about 16 kD (the Ubc domain) which contains the Ubc motif, [FYWLS]-H-[PC]-[NH]-[LIV]-x(3,4)-G-x-[LIV]-C-[LIV]-x-[LIV]. The Ubc domain contains a conserved cysteine residue, which accepts ubiquitin from the ubiquitin-activating enzyme E1 to form a thiol ester. Substitution of the conserved cysteine abolishes E2 activity. A suggested motif rich in basic residues is found at the N-terminus of the UBC domain which may be involved in E1 binding.
E2 enzymes can be classified on the basis of their structure into three classes.
Class I: these proteins comprise simply the “Ubc” catalytic domain. In vitro these enzymes are very poor at transferring ubiquitin to proteins on their own, and probably require an E3 to aid this in vivo. UBC 4 and 5 of S. cerevisiae, UBC1 of Arabidopsis thaliana, and human UBE2D1, UBE2D2, UBE2D3 or UBE2D4 are examples of this class of E2, and are known to be important in the ubiquitination of many short-lived and abnormal proteins prior to degradation.
Class II: these enzymes contain a C-terminal tail attached to the Ubc domain. The tails are different in type but very acidic tails, as found in Ubc2 (also known as Rad6) of S. cerevisiae, appear to mediate inteaction with protein substrates, in this case with the basic histones. Ubc2/Rad6 will ubiquitinate histones in vitro, which requires the C-terminal tail and is known to be involved in DNA repair. This may be a form of ubiquitination that results in protein modification but not degradation. Other C-terminal tails appear to be involved in E2 localisation. Ubc6 of S. cerevisiae is found anchored to the ER membrane with the active site facing the cytosol. The 95 residue C-terminal tail of Ubc6 includes a hydrophobic signal-anchor sequence.
Class III: N-terminal extensions are present in this class of E2s. Several enzymes of this class have been identified but the function of the extensions is unknown.
Ubiquitin binding domains are modular protein elements that bind non-covalently to ubiquitin. They are typically small, being 20 to 150 amino acids in length, and independently-folded, making their isolation straight forward. They are based on a number of different ubiquitin binding motifs. The Ubc of E2 enzymes is one class of ubiquitin binding domain (UBD). Other classes include α-helical domains, zinc finger domains (ZnFs) and plekstrin homology (PH) domains. See, for example, Dikic et al., 2009. Many UBDs are known in the art; for example, see Table 1 in Dikic et al., page 663.
Isopeptidase T (IsoT, or USP5) contains a ZnF-type UBD (known as ZnF UBP or PAZ domain) between amino acid positions 163 and 291 (see Reyes-Turcu et al., 2006). HDAC6 (Boyault et al., 2006) also contains a ZnF UBP domain. Other zinc finger ubiquitin binding domains include UBZ domains, as contained in polymerase-h and polymerase-k; NZF and A20-like ZnF domains.
Alpha-helical types of domains include, for example, UBA domains, found in Rad23 and R23A proteins, or ubiquitin interacting motifs (UIM, MIU or dUIM); see Dikic et al., 2009.
The study of the ubiquitin system requires the ability to produce unattached polymeric ubiquitin in solution, for structural and functional analysis. As noted above, ubiquitin chains vary according to which of the 7 internal Lys residues is used for concatenation of the ubiquitin molecules. In absence of a E3 ubiquitin ligase, most E2 enzymes fail to assemble polyubiquitin. Class II E2 enzymes can assemble polyubiquitin chains on their own C-terminal tails. Very few E2 enzymes, including UBE2R2/cdc34, UBE2K and UBE2S produce free, i.e. unattached, polyubiquitin in solution. For instance, UBE2S, which assembles K-11 linked polyubiquitin, is inefficient at producing free ubiquitin multimers in solution, producing only small amounts of free ubiquitin dimers. There is a need, therefore, for improved E2 enzymes that can be used to produce free polyubiquitin in solution.
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